Biochemical measurement systems are important constituents of clinically relevant methods of analysis. The priority here is to measure analytes, e.g. metabolites or substrates, which are determined directly or indirectly with the aid of an enzyme. The analytes are in this case converted with the aid of an enzyme-coenzyme complex and then quantified. This entails the analyte to be determined being brought into contact with a suitable enzyme and a coenzyme, with the enzyme usually being employed in catalytic amounts. The coenzyme is changed, e.g. oxidized or reduced, by the enzymatic reaction. This process can be detected directly, or electrochemically or photometrically through a mediator. A calibration provides a direct relationship between the measurement and the concentration of the analyte to be determined.
Coenzymes are organic molecules which are bound covalently or non-covalently to an enzyme and which are changed by the conversion of the analyte. Prominent examples of coenzymes are nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, from which NADH and NADPH, respectively, are produced by reduction.
Measurement systems known in the prior art are notable for being stable for a limited period and for the specific requirements on the environment, such as cooling or dry storage, for achieving this stability. For particular applications, e.g. tests carried out by the final user himself, such as, for example, in the self-monitoring of blood glucose, it is therefore possible for incorrect results to occur through incorrect, unnoticed faulty storage. The exhaustion of desiccants through the primary packaging being opened for too long in particular may lead to faulty measurements which, with some systems, can scarcely be identified by the user.
One known measure employed to increase the stability of biochemical measurement systems is the use of stable enzymes, e.g. the use of enzymes from thermophilic organisms. A further possibility is to stabilize enzymes by chemical modification, e.g. crosslinking, or by mutagenesis. In addition, enzyme stabilizers such as, for example, trehalose, polyvinylpyrrolidone and serum albumin can also be added, or the enzymes can be enclosed e.g. by photopolymerization in polymer networks.
Attempts have also been made to improve the stability of biochemical measurement systems by using stable mediators. Thus, the specificity of tests is increased, and interference during the reaction is eliminated, through the use of mediators with a redox potential which is as low as possible. However, the redox potentials of the enzyme/coenzyme complexes form a lower limit for the redox potential of mediators. Below these potentials, the reaction with the mediators is slowed down or even stopped.
An alternative possibility is also to use biochemical measurement systems without mediators, in which for example there is direct detection of coenzymes, e.g. of the coenzyme NADH. One disadvantage of such measurement systems is, however, that coenzymes such as NAD and NADP are unstable.
NAD and NADP are base-labile molecules whose degradation pathways are described in the literature (N. J. Oppenheimer in The Pyridine Nucleotide Coenzymes, Academic Press New York, London 1982, editors J. Everese, B. Anderson, K. You, Chapter 3, pages 56-65). The degradation of NAD and NADP, respectively, essentially results in ADP-ribose through cleavage of the glycosyl linkages between the ribose and the pyridine unit. The reduced forms NADH and NADPH are on the other hand acid-labile: e.g. epimerization is one known degradation pathway. In both cases, the instability of NAD/NADP and NADH/NADPH derives from the lability of the glycosyl linkage between the ribose unit and the pyridine unit. However, even under conditions which are not drastic, such as, for example, in aqueous solution, the coenzymes NAD and NADP, respectively, are hydrolysed solely through the ambient moisture. This instability may lead to inaccuracies in the measurement of analytes.
A number of NAD/NADP derivatives is described for example in B. M. Anderson in The Pyridine Nucleotide Coenzymes, Academic Press New York, London 1982, editors J. Everese, B. Anderson, K. You, Chapter 4. Most of these derivatives are, however, not well accepted by enzymes. The only derivative which has to date therefore been used for diagnostic tests is 3 acetylpyridine adenine dinucleotide (acetyl NAD) which was described for the first time in 1956 (N. O. Kaplan, J. Biol. Chem. (1956), 221, 823). Also this coenzyme shows a poor acceptance by enzymes and a change in the redox potential.
WO 01/94370 describes the use of further NAD derivatives with a modified pyridine group. Modifications of the nicotinamide group have, however, in general a direct influence on the catalytic reaction. In most cases, this influence is negative.
In a further idea for stabilization, the ribose unit has been altered in order thereby to influence the stability of the glycosyl linkage. This procedure does not directly interfere with the catalytic reaction of the nicotinamide group. However, there may be an indirect influence as soon as the enzyme exhibits a strong and specific binding to the ribose unit. Kaufmann et al. disclose in this connection in WO 98/33936 and U.S. Pat. No. 5,801,006, and in WO 01/49247, respectively, a number of thioribose-NAD derivatives. A connection between the modification of the nicotinamide-ribose unit and the activity of the derivatives in enzymatic reactions has, however, not been shown to date.
CarbaNAD, a derivative without a glycosyl linkage, was described for the first time in 1988 (J. T. Slama, Biochemistry 1989, 27, 183 and Biochemistry 1989, 28, 7688). The ribose therein is replaced by a carbocyclic sugar unit. Although carbaNAD was described as a substrate of dehydrogenases, its activity has not to date been demonstrated clinically in biochemical detection methods.
A similar approach was described later by G. M. Blackburn, Chem. Comm., 1996, 2765, in order to prepare carbaNAD with a methylenebisphosphonate compound instead of the natural pyrophosphate. The methylenebisphosphonate shows increased stability towards phosphatases and was used as inhibitor of ADP-ribosyl cyclase. An increase in hydrolysis stability was not the aim (J. T. Slama, G. M. Blackburn).
WO 2007/012494 and U.S. Ser. No. 11/460,366 disclose stable NAD/NADH and NADP/NADPH derivatives, respectively, enzyme complexes of these derivatives and the use thereof in biochemical detection methods and reagent kits.
An object of the present invention is to provide methods for stabilizing enzymes, especially for the long-term stabilization of enzymes.